The adenovirus type 5 i-leader open reading frame functions in cis to reduce the half-life of L1 mRNAs.
The 440-nucleotide adenovirus type 5 i-leader sequence, encoding a 13.6-kilodalton protein, is located between the second and third components of the tripartite leader sequence. It appears primarily on the L1 family of mRNAs. To study its function, we constructed two point mutations within the i leader. pm382 lacks the wild-type i-leader splice acceptor and failed to splice the leader onto L1 mRNAs. pm383 lacks the ATG used for translation of the i-leader protein; it synthesized i-leader-containing mRNAs, but failed to produce detectable levels of the polypeptide. Both mutants exhibited modestly reduced yields in some but not all cell lines tested and accumulated slightly elevated levels of L1 mRNA and L1 52- and 55-kilodalton proteins in infected cells. Mutant phenotypes were consistently more pronounced in pm382- than in pm383-infected cells. In wild-type virus-infected cells, L1 mRNAs lacking the i leader displayed a half-life of about 26 h, whereas L1 mRNAs containing the leader were much less stable, with a half-life of less than 4 h. In pm383-infected cells (ATG mutant), L1 mRNAs containing the i leader exhibited a half-life of 26 h. The abnormally long half-life of pm383-encoded L1 mRNAs containing a mutant i leader was not reduced by coinfection with wild-type virus, suggesting that synthesis of the i-leader protein leads to destabilization of the i-leader-containing L1 mRNA undergoing translation.
Soloway
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Howard Hughes Medical Institute, Department of Biology, Princeton University, New Jersey 08544.
Shenk
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D000260:Adenoviruses, Human / Q000235:genetics* / Q000254:growth & development; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003609:Dactinomycin / Q000494:pharmacology; D005814:Genes, Viral; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014709:Vero Cells
D015345:Oligonucleotide Probes; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D003609:Dactinomycin; D008715:Methionine